One of the major elements found in nearly all complex macromolecules of living cells is nitrogen. Nitrogen is particularly important for synthesis of proteins and nucleic acids and it is not surprising that cells possess major control mechanisms to regulate nitrogen metabolism and ensure a constant adequate supply of this nutrient for growth. The experiments outlined in this proposal are designed to investigate, at the molecular level, regulation of nitrogen metabolism in the yeast Saccharomyces cerevisiae. Yeast provides an excellent model system for these studies because of the ease of both biochemical analysis and advanced molecular genetics in this organism. Yeast mutants have been isolated that are unable to growth on rich media (YPD) but grow normally on minimal media using ammonia as nitrogen source. Addition of single amino acids (phenylalanine, threonine, serine, isoleucine, valine, glutamate, glutamine) at low concentrations is toxic to these mutants growing on minimal ammonia plates. However, they grow normally on poor nitrogen sources (e.g., proline) and are unaffected by addition of these amino acids. These results suggest the YPD- phenotype has been caused by an imbalance of nitrogen metabolism in these strains and I have named them AAT (amino acid toxicity) 1 and AAT2. A complete biochemical analysis of nitrogen metabolism in aat yeast strains is proposed in an attempt to identify the specific lesion in these mutants. In particular, the levels of key enzymes (e.g., glutamate dehydrogenase), amino acid pools and amino acid transport will be determined. Regulation of these reactions will be studied to determine the level of nitrogen repression in these strains. I also propose to start a genetic analysis of these strains. Clones have been isolated that complement the YPD- phenotype of both aat1 and aat2 yeast. These will be further characterized and then used to obtain information about the AAT genes and the proteins they encode by the techniques of yeast molecular genetics.